The helix-loop-helix (HLH) motif has been found in many genes required for the processes of sex-determination, segmentation, neurogenesis, and myogenesis in Drosophila, as well as during neurogenesis and myogenesis in mammals. A functional relationship between HLH proteins and oncology has also been demonstrated by the over-expression of the myc HLH proto-oncogene in many cancers and the involvement of HLH genes in acute T-cell leukemia. In many cases, these HLH proteins are involved in developmental "switch" decisions: they regulate the choice between alternate pathways and effect changes in cell fate. The HLH motif mediates protein dimerization, that in turn mediates sequence-specific DNA binding. However, the rules that govern the specificity of interactions among these factors and their outcomes are largely unknown. The longterm goal of the proposed research is to understand the mechanisms by which these proteins regulate transcription and orchestrate specific cell determinations. This study will use genetic, developmental, and molecular genetic approaches in Drosophila to investigate both the function of specific HLH proteins and the rules that govern HLH protein function in general. My earlier work with the HLH-containing segmentation gene hairy led to an identification of HLH proteins involved in sex-determination and provides an in vivo model system for many of these approaches. The specific aims of this proposal are: (1) analysis of the requirements for HLH proteins in sex-determination; (2) investigation of the specificity of related HLH proteins; (3) analysis of the structural and functional properties of the hairy HLH transcriptional regulator; and (4) identification of genes interacting with or regulating the hairy gene during segmentation. The numerous processes requiring HLH proteins and the wealth of techniques/reagents available in Drosophila make it an excellent organism for studying the functions of HLH proteins in vivo. Results obtained from these projects are expected to complement the in vitro dimerization/binding studies being conducted in other labs.